TRANS-SPLICING IN C ELEGANS

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DESCRIPTION (Adapted from Applicant's Abstract): Nuclear mRNA processing is catalyzed by spliceosomes which are composed of small nuclear ribonucleoprotein particles (snRNPs) and additional proteins. In C. elegans this process is essentially similar to splicing in higher eucaryotes, except the introns tend to be much smaller. Nematodes also perform an alternative spliceosome-catalyzed mRNA splicing event, trans- splicing, in which the RNA products of two separate gene are splices together to form a single mature mRNA. While some genes are conventional in that they initiate transcription at the 5' end of exon 1, the pre mRNA products of others receive a 22 nucleotide exon 1 by trans-splicing. This exon is donated by a 100 nucleotide RNA molecule, SL RNA, which is itself packaged as a snRNA. C. elegans has two distinct SL snrnps, each of which delivers its 5' 22 nucleotides to a distinct group of pre-mRNAS. The investigator is exploring the relationship between normal (cis-) splicing and trans-splicing. Why are the pre-mRNA products of some genes recipients for SL RNA splicing which others are not; Where do the specificity determinants for SL1 vs. SL2 splicing reside, both on the SL snRNP and on the recipient pre-mRNAs? The investigator has shown that, when moved into an appropriate context, an intron 3' splice site is sufficient to convert a conventional gene into a trans-spliced gene. Conversely the investigator has shown that it is possible to convert a trans-spliced gene into a conventional gene by inserting a 5' splice site upstream of its trans- splicing site. In the current application the investigator proposes a series of transformation experiments in which genes are mutated, introduced into worms, and tested for the splicing events they undergo. The following specific questions will be answered; (I). What are the branch point and 3' splice site sequence requirements for trans-splicing? (II). How is the trans-splice site chosen? (III). Is there a minimal or maximal outron length? (The investigator defines the region of a trans-spliced transcript between its 5' end and the trans-splice site as an "outron." (IV). Is an overall high percent A+U in the outron required for trans-splicing? (V). What factors influence the competition that occurs between cis-and trans- splicing when a 5' splice site is present in an outron? In this situation how critical is distance of the 5' splice site from the trans-splice site? (VI). Where is the information on the recipient RNA that results in specific splicing of SL1 or SL2? (VII). Where is the information on SL1 RNA and SL2 RNA for specific trans-splicing?